![]() As a result, there is no unobstructed path by which dsDNA can access the RNAP active-centre cleft in RNAP holoenzyme ( Mekler et al., 2002 Murakami et al., 2002b Murakami et al., 2002a Vassylyev et al., 2002 Young et al., 2002 Murakami and Darst, 2003). In crystal structures of RNAP σ holoenzyme, the RNAP active-centre cleft is too narrow to accommodate double-stranded DNA (dsDNA) (<20 Å), and σ obstructs access of dsDNA to the RNAP active-centre cleft ( Mekler et al., 2002 Murakami et al., 2002b Murakami et al., 2002a Vassylyev et al., 2002). However, the mechanism by which DNA is unwound and loaded into the RNAP active-centre cleft has remained controversial (reviewed in Mazumder and Kapanidis, 2019). Structural and biochemical experiments suggest that transcription-bubble formation is initiated by unwinding the DNA base pair at the upstream end of the transcription bubble – breaking the base pair, and unstacking and flipping the non-template-strand base of the broken base pair, and inserting the unstacked and flipped non-template-strand base into a protein pocket of σ – followed by propagation of the unwinding in a downstream direction ( Mazumder and Kapanidis, 2019 Ruff et al., 2015 Zhang et al., 2012 Feklistov and Darst, 2011). High-resolution structures of RPo define the contacts that RNAP and σ make with promoter DNA, as well as the conformation and interactions of unwound template-strand ssDNA engaging the RNAP active centre ( Bae et al., 2015 Zuo and Steitz, 2015 Zhang et al., 2012). During transcription initiation, RNA polymerase (RNAP), together with the transcription initiation factor σ, unwinds ~13 bp of promoter DNA to form a ‘transcription bubble’, and places the template-strand ssDNA of the unwound transcription bubble in contact with the RNAP active centre, yielding a catalytically competent RNAP-promoter transcription-initiation complex (RPo Mazumder and Kapanidis, 2019). Transcription initiation is the first and most highly regulated step in gene expression ( Mazumder and Kapanidis, 2019 Ruff et al., 2015). Our work defines a new, ‘bind-unwind-load-and-lock’, model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyse RPo formation at a consensus bacterial core promoter. Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. ![]() ![]() Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPo). ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |